Surface Phenotyping
Flow cytometric characterisation of T cells was carried out with surface marker panel: CD45RA-Vioblue, CD8-Viogreen, CD62L-FITC, CD3-PE or CD57-PE, CD45RO-PE-Vio700, CD4-PE-Vio770, CD28-APC (all Miltenyi Biotech) and dead cell marker DRAQ7 (BioLegend, UK). Following stimulation by LCL, samples were taken weekly to monitor phenotypic changes using a modified panel, where CD57-PE (Miltenyi) was used to replace CD3 to monitor terminal differentiation / senescence.
Briefly, 2x106 cells were washed with PBS plus 2.5mM EDTA (Invitrogen) and 0.5% human serum albumin (Alburex) (PEA buffer), blocked with 5μl human Fc Receptor block (Miltenyi), labelled with antibody cocktail at 4°C for 20 minutes, washed and DRAQ7 added prior to analysis. Approximately 100,000 live events were acquired using a MACSQuant10 (Miltenyi) or BD Fortessa LSR (Becton Dickinson) flow cytometer with data analysis using FlowJo V10 software (Tree Star, Inc). All flow cytometric analyses were based on a manual gating strategy as described in Figure 1A, with lymphocyte population identified by scatter, then doublets and dead cells excluded (singlet gated / DRAQ7- cells). Data were collected as percentages of gated populations, as mean fluorescence intensity (MFI), or as a corrected MFI (target MFI – isotype MFI).